Targeting the NRF2 pathway for disease modification in neurodegenerative diseases: mechanisms and therapeutic implications

Neurodegenerative diseases constitute a global health issue and a major economic burden. They significantly impair both cognitive and motor functions, and their prevalence is expected to rise due to ageing societies and continuous population growth. Conventional therapies provide symptomatic relief, nevertheless, disease-modifying treatments that reduce or halt neuron death and malfunction are still largely unavailable. Amongst the common hallmarks of neurodegenerative diseases are protein aggregation, oxidative stress, neuroinflammation and mitochondrial dysfunction. Transcription factor nuclear factor-erythroid 2-related factor 2 (NRF2) constitutes a central regulator of cellular defense mechanisms, including the regulation of antioxidant, anti-inflammatory and mitochondrial pathways, making it a highly attractive therapeutic target for disease modification in neurodegenerative disorders. Here, we describe the role of NRF2 in the common hallmarks of neurodegeneration, review the current pharmacological interventions and their challenges in activating the NRF2 pathway, and present alternative therapeutic approaches for disease modification.


Introduction
Neurodegenerative diseases are a heterogenous group of neurological disorders characterized by the progressive loss of selective neuronal populations in the nervous system, giving rise to distinct clinical features, such as cognitive, behavioral, and motor symptoms (Agrawal, 2020).Amongst the most common neurodegenerative diseases are Alzheimer's disease (AD), Parkinson's disease (PD), Multiple Sclerosis (MS) and Amyotrophic Lateral Sclerosis (ALS).Importantly, the burden of neurodegenerative diseases on the healthcare system is projected to increase globally in the coming decades, due to the continuous population growth, an ageing society and the lack of early diagnostic biomarkers and effective medical treatments (Peplow et al., 2022).Despite their distinct clinical features and underlying pathophysiology, neurodegenerative diseases share fundamental disease processes associated with neuronal dysfunction and death (Erkkinen et al., 2018).Of those pathological characteristics, aberrant protein deposition, oxidative stress, neuroinflammation and mitochondrial dysfunction are believed to constitute the main molecular hallmarks (Wilson et al., 2023;Kovacs, 2019).
Transcription factor nuclear factor-erythroid 2-related factor 2 (NRF2) is a central regulator of the cellular defense line against toxic and oxidative insults.Its activation upregulates antioxidant defenses, inhibits inflammation, improves mitochondrial function, and maintains protein homeostasis (Cuadrado et al., 2019).As a member of the Cap 'n' Collar (CNC) transcription factor family, NRF2 exerts its function in the nucleus, by forming heterodimers with small musculoaponeurotic fibrosarcoma proteins (sMAFs), enabling NRF2 to bind to the enhancer region of the antioxidant response elements (AREs), which function as regulatory DNA sequences associated with the transcriptional activation of antioxidant enzymes and regulation of redox homeostasis (Katsuoka and Yamamoto, 2016).
NRF2 protein stability is tightly regulated and its two main regulators are i) Kelch-like ECH-associated protein 1 (KEAP1) in the cytoplasm and ii) BTB and CNC homology 1 (BACH1) in the nucleus.Under physiological conditions, KEAP1 mediates NRF2s constitutive proteasomal degradation (McMahon et al., 2003) while BACH1 acts as transcriptional antagonist of NRF2s target genes (Dhakshinamoorthy et al., 2005).During oxidative stress conditions, KEAP1 is inactivated by chemical modifications at its reactive cysteine residues, which allows NRF2 to escape degradation and for newly synthesized NRF2 to translocate to the nucleus (Zhang and Hannink, 2003).In turn, oxidative stress insults upregulate heme, BACH1s ligand and mediator for nuclear export, allowing for gene induction of NRF2-regulated cytoprotective factors (Ogawa et al., 2001;Reichard et al., 2007) (Figure 1).
In this article, we will review how NRF2 activity is associated with neurodegenerative diseases, particularly with disease hallmarks such as oxidative stress, neuroinflammation and mitochondrial dysfunction.We will then discuss the validity of the most common molecular targets; the cytoplasmic inhibitor KEAP1 and the transcriptional repressor BACH1, together with the different drug modalities serving as NRF2-activators.Finally, we will review the use of novel therapeutic approaches for activation of NRF2 as a disease-modifying treatment for neurodegenerative diseases.

NRF2 coordinates cellular defense processes against diverse pathological hallmarks of neurodegeneration
Neurodegenerative diseases are characterized by common pathological hallmarks such as oxidative stress, neuroinflammation, mitochondrial dysfunction and protein aggregation.Targeting these features simultaneously holds the potential to achieve true disease modification, as it addresses the complexity and multifactorial nature of neurodegeneration.In accordance, targeting of NRF2 is of high interest, due to its prominent roles in several neurodegenerative hallmarks.
Neuroinflammation: NRF2 has been shown to be a central regulator of neuroinflammation (Saha et al., 2021).Mechanistically, it can dampen the pro-inflammatory response by decreasing the transcription of pro-inflammatory cytokines through direct DNA binding to Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (Wang and He, 2022).In fact, these antiinflammatory effects have been demonstrated in mouse microglia, macrophages, monocytes, and astrocytes (Kobayashi et al., 2016;Quinti et al., 2017).
Mitochondrial dysfunction: NRF2 also orchestrates mitochondrial proteins such as superoxide mutase 1 (SOD1), which scavenges mitochondrial ROS and RNS to protect the mitochondrial respiratory chain from oxidative insults (Cassina et al., 2008;Abdalkader et al., 2018), PTEN-induced putative kinase 1 (PINK1), a protein crucially important for sustaining functional mitochondrial homeostasis (Gumeni et al., 2021) and protein deglycase DJ-1 (PARK7), which maintains mitochondrial complex I activity and the mitochondrial membrane potential, two critical indicators for mitochondrial health (Hayashi et al., 2009;Im et al., 2012).Activation of NRF2-mediated antioxidant enzymes has been shown to enhance the biogenesis of mitochondria in mice and humans (Piantadosi et al., 2008;Merry et al., 2016;Hayashi et al., 2017) and by improving mitochondrial function, the excessive production of ROS was mitigated in mouse embryonic fibroblasts (Kovac et al., 2015).
Despite being considered as distinct pathological events, oxidative stress, neuroinflammation and mitochondrial dysfunction interplay with and enhance each other (Picca et al., 2020) (Figure 2).The main intracellular producer of ROS and RNS is the mitochondrial respiratory chain (Ray et al., 2012) and the increase of oxidative damage or release of mitochondrial DNA observed during mitochondrial dysfunction act as dangerassociated signals (DAMPs), which initiate the immune response through pro-inflammatory NF-κB activation (Picca et al., 2020).While immune activation can effectively clear damaged mitochondria (Harding et al., 2023), immune dysfunction has been reported to trigger mitochondrial impairments and thereby increase the oxidative damage within the cell (Tresse et al., 2021).Taken together, the above findings illustrate the importance of the complex relationship between oxidative metabolism, inflammation, and neurodegeneration.

Role of NRF2 in neurodegenerative diseases
Oxidative stress, neuroinflammation, and mitochondrial dysfunction are prevalent characteristics of the aging brain and research indicates that the expression of NRF2 and its downstream genes declines with age (Schmidlin et al., 2019).In fact, aging stands out as the main risk factor for the development and progression of neurodegenerative diseases (Hindle, 2010).Hence, NRF2 has emerged as an attractive target for the clinical intervention of neurodegeneration and in the following section, we describe the role of NRF2 in AD, PD, MS and ALS.

NRF2 in Alzheimer's disease
AD is the most common neurodegenerative disease and contributes to 60%-70% of the 55 million people diagnosed with dementia worldwide (WHO, 2023).Clinically, AD is characterized by progressive cognitive decline and pathologically, its two main hallmarks are intracellular neurofibrillary tangles comprised of the protein tau and extracellular β-amyloid (Aβ) plaques (Deture and Dickson, 2019).Emerging evidence suggests that Aβ plaques contribute synergistically to disease progression together with oxidative stress, inflammation and mitochondrial dysfunction, while this is less clear in regards to tau pathology (Brackhan et al., 2023).Studies have reported that Aβ plaques are tightly linked to oxidative stress (Allan Butterfield and Boyd-Kimball, 2018;Roy et al., 2023), since Aβ aggregation has been determined as source of oxidative stress (Karapetyan et al., 2022) and oxidative stress has been shown to increase the aggregation of Aβ (Mandal et al., 2022).Aβ plaques have also been associated with neuroinflammation (Leng and Edison, 2020;Sobue et al., 2023) and activated microglia has been reported to surround the protein depositions (Heneka et al., 2013), contributing to the spread of Aβ plaques (d'Errico et al., 2021) leading to an increase in pro-inflammatory cytokines (Rani et al., 2023).Additionally, Aβ has been observed to localize to mitochondria and to produce free radicals impairing the bioenergetic machinery (Manczak et al., 2004;2006), while gene expression studies have identified a significant downregulation of genes related to the oxidative phosphorylation system in AD brains (Mastroeni et al., 2017).
In the AD brain, NRF2 has been mainly found within the cytoplasm and its nuclear fractions significantly reduced compared to age-matched healthy controls (Ramsey et al., 2007).Interestingly, the reduction of NRF2 levels has been associated with increased production of Aβ (Bahn et al., 2019).Consistent with these observations, molecular pathways that are known to be altered in AD brains, including severe amyloidopathy, tauopathy and Synergistic interplay and dynamic relationships between neurodegenerative diseases hallmarks contribute to neurodegeneration.Mitochondrial dysfunction, oxidative stress and neuroinflammation reciprocally influence the progressive loss of structure and function in neurons in diseased brains.While damaged mitochondria significantly contribute to the increased generation of ROS and RNS, leading to oxidative stress which damages surrounding biomolecules, those reactive species and the release of mtDNA also act as danger signals activating the immune response.exacerbated cognitive defects, were found dysfunctional in NRF2 knockout mice too (Rojo et al., 2017).
Conversely, NRF2 activation has been shown to exert beneficial effects in diverse disease models in vitro and in vivo.In AD astrocytes derived from pluripotent stem cells pharmacological activation of NRF2 ameliorated amyloid secretion and reduced inflammatory cytokine expression (Oksanen et al., 2020).In an AD mouse model (APP NLGF ), increased expression of NRF2 reduced inflammation and oxidative stress and improved cognition (Uruno et al., 2020).Furthermore, several small molecules have been shown to activate the NRF2/ARE pathway and decrease Aβ pathology in vitro and in vivo (Ikram et al., 2019;Fakhri et al., 2020).In a clinical setting, therapies specifically targeting NRF2 have yet to be investigated, nevertheless, there is some evidence that treatment with antioxidant compounds could be beneficial.For instance, a clinical trial using Vitamin E as NRF2 activator revealed slowed decline in mini-mental state exam and activities of daily life (Dysken et al., 2014).

NRF2 in Parkinson's disease
PD constitutes the most common movement disorder, affecting over 8.5 million people worldwide (Ding et al., 2022).PD is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) as well as the presence of α-synuclein-containing protein aggregates in the brain, referred to as Lewy bodies (Morris et al., 2024).Oxidative stress, dysfunctional mitochondria, and neuroinflammation have all been suggested to play roles in the onset and progression of PD (Navarro and Boveris, 2009;Di Filippo et al., 2010).
The presence of oxidative stress has been shown by of increased levels of oxidative damage to lipids, protein, and DNA in the parkinsonian brain (Alam et al., 1997;Mythri et al., 2011).These oxidative modifications can further activate microglia through DAMP-associated pathways and interestingly, α-synuclein misfolding has also been associated with microglia activation (Béraud and Maguire-Zeiss, 2012).Stimulation of the immune response has been demonstrated in various post-mortem studies, which found elevated levels of pro-inflammatory cytokines (Nagatsu et al., 2000;Brodacki et al., 2008;Reale et al., 2009) and additional evidence suggests that several proteins, which are highly expressed by microglia and astrocytes, such as Leucine-rich repeat kinase 2 (LRRK2), PINK1 and PARKIN, are encoded by genes implicated in familial forms of PD (Scarffe et al., 2014;Brockmann et al., 2016;Isik et al., 2023) Furthermore, excessive production of ROS has also been linked to dysfunctions in mitochondrial complex I and the complex' activity reported to be reduced in PD patient brains (Keeney et al., 2006).Coherently, complex I inhibitors like the neurotoxin 1methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) lead to PDlike symptoms in humans and animals, accompanied by the loss of adenosine triphosphate (ATP) production and increased generation of oxidative stress (Smith and Bennett, 1997).Additionally, mtDNA damage has been associated with PD etiology and progression (Tresse et al., 2023) and mitochondrial dysfunction is linked genetically to PD, since many mutations associated to familial cases of PD were found in genes such as PINK1 or PARKIN, which regulate mitochondrial turnover (Valente et al., 2004;Lev et al., 2006;2008;Paterna et al., 2007;Devi et al., 2008).
Failure of the NRF2 pathway has been reported in PD.Pathway dysregulation has been observed in the brain, urine and plasma of PD patients, showing a significant reduction of antioxidant enzyme activity along with an increased level of oxidative stress (Colamartino et al., 2018).Also, mouse models of PD have indicated decreased NRF2 activity in astrocytes (Chen et al., 2009) and knockdown of NRF2 has been shown to cause an increase in mitochondrial ROS production (Kovac et al., 2015), the loss of dopaminergic neurons in association with microglia activation (Rojo et al., 2010) and exacerbation of synuclein aggregation (Lastres-Becker et al., 2012).
Importantly, NRF2 activation has been shown to alleviate PDassociated pathological hallmarks, specifically oxidative stress, mitochondrial dysfunction and neuroinflammation through expression of its target genes (Yang et al., 2022).In the MPTP mouse model, NRF2 activation has been associated with a reduction in oxidative stress (Williamson et al., 2012), induction of antioxidant enzymes (Jazwa et al., 2011), and improvement of the mitochondrial respiratory rate (Fu et al., 2018).Anti-inflammatory properties of NRF2 signaling have been verified in LPS-stimulated mouse microglia, where its upregulation reduced inflammatory markers (Townsend and Johnson, 2016) and astrocytic, and microglial activation were found to be significantly lower (Jing et al., 2015).Additionally, treatment with NRF2 activators in PINK1-or PARKIN-deficient drosophila restored mitochondrial function, elevated mitophagy rates and suppressed oxidative stress (Gumeni et al., 2021).In conclusion, these data unveil the therapeutic potential of increasing NRF2 signaling for treating PD.

NRF2 in Multiple Sclerosis
MS is a chronic neurodegenerative and autoimmune disease that affects 2.8 million people worldwide and remains one of the most common causes of neurological disability in the young adult population (Jakimovski et al., 2024).Its main pathological hallmark constitutes demyelinating lesions that accumulate within the central nervous system (CNS) (Popescu et al., 2013;Filippi et al., 2018) leading to cognitive and motor disability.The demyelinating process is accompanied, amongst other factors, by extensive neuroinflammation (Gold et al., 2006).Elevated levels of proinflammatory cytokines (Khaibullin et al., 2017) and increased levels of chemokines responsible for recruiting peripheral immune cells and activating the adaptive immune response (Kalinowska-Łyszczarz et al., 2011), have been found in MS patients.Additionally, altered expression of genes involved in the activation of the adaptive immune response have been observed and provide the genetic link between MS and progressive neuroinflammation (Ramanathan et al., 2001).The prolonged activation of macrophages and microglia has been associated with the increased production of ROS (Hemmati et al., 2020) and eventually results in oxidative stress (Zhang et al., 2020).This has been demonstrated in MS patients as increased concentrations of ROS (Acar et al., 2003;Gilgun-Sherki et al., 2004) along with decreased antioxidant levels (Choi et al., 2018;De Riccardis et al., 2018) were found in serum and cerebrospinal fluid.The abundant generation of ROS along with the enhanced neuroinflammatory response then negatively affect mitochondrial function (Forte et al., 2007;Blagov et al., 2022).As observed in human studies, where mitochondrial oxidative phosphorylation complexes were decreased in the MS cortex (Witte et al., 2013) together with mutated mtDNA and reduced mtDNA copy numbers (Ban et al., 2008;Blokhin et al., 2008).
The role of NRF2 on MS pathogenesis becomes observable in both disease models as well as patients.The experimental autoimmune encephalomyelitis (EAE) or lipopolysaccharide (LPS)-induced MS effectively recapitulate the inflammatory response, mitochondrial dysfunction, and oxidative stress (Wang et al., 2017;Yang et al., 2018).In those, loss of NRF2 resulted in a more rapid onset and more severe clinical course of the EAE model, which was accompanied by increased glial activation, exacerbated spinal cord damage, and axonal degeneration as well as increased levels of proinflammatory cytokines (Johnson et al., 2010;Larabee et al., 2016).Additionally, NRF2-activators have shown beneficial effects in MS model systems.These effects include the reduction of LPS-induced neurotoxicity, lowering inflammatory markers and gliosis, and improvement of synaptic and mitochondrial function (Larabee et al., 2016;Chen et al., 2017;Khan et al., 2018;Rehman et al., 2019).
In fact, NRF2 activation as a clinical target is well established for the treatment of MS since the potent NRF2-activator dimethyl fumarate (DMF) was approved by FDA as first disease-modifying agent in 2013 (Fox et al., 2012;Gold et al., 2012).DMF has been shown to alleviate hallmarks of neurodegenerative diseases through the blockage of pro-inflammatory NF-κB (Gillard et al., 2015), the upregulation of the antioxidant NRF2-ARE pathway (Rosito et al., 2020), and by increasing mitochondrial function (Hui et al., 2021).Importantly, MS patients who showed a significant increase in the expression of NRF2's downstream target, NQO-1, following DMF treatment were more likely to achieve no evidence of disease activity after 1 year (Hammer et al., 2018).

NRF2 in Amyotrophic Lateral Sclerosis
ALS is the most common motor neuron disease in adults and estimates have reported that 31,000 patients live with ALS in the US, where on average 5,000 new patients are diagnosed annually (Mehta et al., 2023).ALS is characterized by neurodegeneration of motor neurons in brain, spinal cord, and periphery, leading to progressive muscle paralysis: terminal events often comprise aspiration pneumonia and respiratory insufficiency (Morgan and Orrell, 2016;Taylor et al., 2016).Approximately 90%-95% of ALS cases appear sporadically, whereas ca.5%-10% show a familial predisposition (Andersen and Al-Chalabi, 2011).The most frequent genetic mutations and defects are found in SOD1 (Ghasemi and Brown, 2018), a gene encoding an essential copper/zinc antioxidant enzyme which is likely involved in both familiar and sporadic presentations (Tafuri et al., 2015).
Notably, mutations and defects in SOD1 have been associated with oxidative stress, neuroinflammation and mitochondrial dysfunction.Increased oxidative damage has been detected in both sporadic and familial ALS patients carrying SOD1 mutations (Abe et al., 1995;Beal et al., 1997;Blasco et al., 2017).Post-mortem studies have described increased activation of microglia and astrogliosis (Chiot et al., 2020) and elevated levels of inflammatory cytokines have been reported in ALS patient's serum (Tortelli et al., 2020).Being a mitochondrial antioxidant enzyme, mutated SOD1 has further been observed to aggregate at the outer mitochondrial membrane and is thought to lead to mitochondrial damage including morphological changes, excessive superoxide production, increase in mitochondria membrane permeability and defective mitophagy (Higgins et al., 2003;Tak et al., 2020).
Importantly, mutant SOD1 has been demonstrated to decrease the expression of antioxidant transcription factor NRF2 and its dependent target genes in an established NSC134 cellular model for SOD1-associated ALS (Kirby et al., 2005).In post-mortem patient samples, both messenger RNA (mRNA) and protein levels of NRF2 have been reported to be reduced (Sarlette et al., 2008) and proteomic analysis of spinal motor neurons from humans with SOD1-related ALS had revealed that the NRF2-induced gene peroxiredoxin is transcriptionally repressed (Wood-Allum et al., 2006).It is important to note that a recent study has demonstrated a significant upregulation of NRF2, HMOX-1 and NQO-1 mRNA expression, as well as an increase in HMOX-1 and NQO-1 protein levels in the spinal cord of ALS patients (Lastres-Becker et al., 2022).However, their results have also demonstrated increased oxidation of lipid substrates, indicating elevated oxidative stress levels (Lastres-Becker et al., 2022), which suggests that the NRF2-mediated antioxidant machinery may be activated but to an insufficient degree in ALS.
In support of the NRF2 pathway being a potential target for the treatment of ALS, studies have shown that NRF2 overexpression in astrocytes provided neuroprotection against mutated SOD1induced toxicity and increased the median survival of mutant SOD1 transgenic mice (Vargas et al., 2008;2013).In vitro studies have demonstrated that overexpression of Angiogenin, a RNase associated with familial ALS, activates NRF2 which protects astrocytes from oxidative stress (Hoang et al., 2019) and curcumin mediated NRF2 induction ameliorates mitochondrial dysfunction (Lu et al., 2012).Further evidence of NRF2s beneficial effects on ALS pathology were reported by clinical studies, which have shown that pharmacological activation of NRF2 improved the disease progression of ALS patients and reduced oxidative stress (Ahmadi et al., 2018;Chico et al., 2018).
Collectively, these data show that the NRF2 pathway is dysfunctional in various neurodegenerative diseases and the effects of the reduced or lost activity of the NRF2-mediated antioxidant pathway in the brain are summarized in Figure 3. Furthermore, those findings suggest that the NRF2 pathway plays an important role in protecting neuronal function as well as in preventing cognitive decline through regulation of a multitude of pathological pathways implicated in neurodegenerative diseases.Thus, targeting the NRF2 pathway holds great potential for being a disease-modifying treatment.

Targeting the NRF2 pathway for disease modification in neurodegenerative diseases
To date, conventional treatment options for neurodegenerative diseases only provide symptomatic relief, and do not interfere with the underlying neuropathological processes.The majority of clinical trials investigating disease-modifying therapies have sought to halt or reverse the deposition of specific proteins in the brain and, while the first disease-modifying drug targeting protein deposition has been approved for AD (Cummings, 2023), two different phase II clinical trials of antibodies targeting α-synuclein in PD patients have not shown any effect on symptom relief (Kharel and Ojha, 2023).Unfavorably, when these protein inclusions are diagnosed, clinical manifestations have started to appear, and underlying pathological hallmarks are greatly advanced (Kalia and Lang, 2015;Ower et al., 2018), which hinders early and successful therapeutic intervention.Furthermore, neurodegenerative diseases develop from a multitude of pathological hallmarks, hence, the therapeutic targeting of only one of them might not have a significant effect on the course of a heterogenous disorder.
Developing effective drugs that can modify the progression of neurodegenerative diseases is crucial and would represent a major medical breakthrough.Due to their complexity, unclarified pathogenesis, progressive and severe nature, it is likely that a truly disease-modifying therapy for neurodegenerative diseases needs to regulate several pathological pathways simultaneously (Li et al., 2020).Hence, pharmaceuticals that target the NRF2 pathway can influence multiple pathways involved in the neurodegenerative process, and therefore hold potential to become therapies that can significantly modify the course of neurodegenerative diseases.
Numerous compounds that activate NRF2 through various mechanisms and structural compositions have been identified (Amoroso et al., 2023).NRF2 activators include naturally occurring molecules such as phytocompounds and cannabinoids, semisynthetic molecules such as Omaveloxolone, or dimethyl fumarate (DMF), the longest NRF2 activator in clinical use (Bomprezzi, 2015).Herein, we discuss the credibility and efficacy of the most prevalent class of NRF2 activators, namely, KEAP1 inhibitors, while also introducing a novel focal point within the NRF2 pathway: the transcription factor BACH1.

Pharmacological activation of NRF2: KEAP1-inhibitors
Thus far, the most common class of NRF2-activators mimic the inhibition of NRF2s cytoplasmic negative regulator KEAP1, an adaptor protein for the CUL3/RBX1 E3 ubiquitin ligase complex, to prevent NRF2 proteasomal degradation (Kobayashi et al., 2004).With regards to neurodegenerative diseases, the two following described compounds, DMF and Sulforaphane (SFN), are amongst the most well-studied compounds of the KEAP1inhibitory category.Nevertheless, it is worth mentioning that other compounds, such as Curcumin (Shahcheraghi et al., 2022), Resveratrol (Yadav et al., 2022) or Ursodiol (Huang, 2021) also have been reported to exert activating effects on NRF2 in neurodegenerative diseases.
Dimethyl Fumarate (DMF, Tecifidera ® ) is an NRF2 activator in clinical use for the treatment of relapsing forms of MS and psoriasis.DMF has been shown to alleviate hallmarks of neurodegenerative Summary of the effects of reduced activity or loss of NRF2 in the brain.Loss or reduced activity of NRF2 enhances oxidative stress, mitochondrial dysfunction and neuroinflammation, which altogether contribute to neurodegeneration.Impaired NRF2 signaling was associated with a decrease in antioxidant enzyme activity, which is responsible for the incapability of cells to remove oxidative insults and therefore results in higher exposure to oxidative stress.Mitochondria are both a major source, and immediate target of oxidative stress.Reduced mitochondrial membrane potential hinders the production of chemical energy ATP, and simultaneously, increases the generation of ROS and RNS, rendering the enzymes responsible for eliminating those reactive species inefficient.Discarded mtDNA can then act as DAMP and trigger danger-signal receptors in microglia, which will further activate astrocytes and together, they induce a broad pro-inflammatory cytokine response through transcription factor NF-κB. Prolonged immune activation disrupts the blood brain barrier (BBB) permeability and allows more immune cells to infiltrate and intensify the immune reaction.Cooperatively, these three pathological hallmarks count, amongst others, as causes of neurodegeneration, which comprises neuronal damage and decline, including dysfunctional neuronal signaling, and aberrant protein aggregation.
diseases such as inflammation and oxidative stress through the blockage of pro-inflammatory NFkB (Gillard et al., 2015) and upregulation of the antioxidant NRF2-ARE pathway (Rosito et al., 2020).This drug has been demonstrated to exhibit several distinct modes of actions, one of them being the irreversible modulation of KEAP1 active cysteine residues (Piroli et al., 2019).Furthermore, studies have shown that DMF causes BACH1 nuclear exit in rat dopaminergic neurons and human cells (Ahuja et al., 2022) (Table 1).These observations indicate that DMFs activation of NRF2 originates not only from inducing NRF2 in the cytoplasm but also from inhibiting the NRF2 repressor in the nucleus.In SH-SY5Y cells stimulated with Aβ 1-42 or 6-OHDA, DMF has been reported to increase cellular viability and reduce intracellular ROS production, both through the activation of HMOX-1, while a study treating ALS patient-derived macrophages demonstrated DMFs ability to inhibit pro-inflammatory cytokines as well as NF-kB (Table 1).Furthermore, AD and PD studies in mice found higher levels of antioxidant enzymes and a reduction of inflammatory markers in the brain, as well as improvement of behavioral impairment upon treatment with DMF (Table 1).Collectively, these data show that DMF constitutes an attractive compound for the pharmaceutical industry for drug repurposing in other neurodegenerative diseases (Bresciani et al., 2023).
Sulforaphane was the first identified and most potent naturally occurring NRF2 activator found in cruciferous vegetables such as broccoli, cauliflower, and Brussel sprouts (Zhang et al., 1992;Yagishita et al., 2019).SFN has been shown to exert antioxidant, anti-inflammatory and antiapoptotic effects through suppression of NRF2's physiological inhibitors KEAP1 and Glycogen synthase kinase-3 beta (GSK-3β) (Zheng et al., 2022).In combination with its excellent bioavailability in the central nervous system, these properties have led to the compound gaining interest from the scientific community for the treatment of neurodegenerative diseases.In vitro models of AD, PD and MS have demonstrated that SFN is efficient in counteracting cell death, neuroinflammation, oxidative stress and protein-aggregation induced by various toxins such as A? plaques, LPS, 6-OHDA or hydrogen peroxide, through upregulation of the NRF2/ARE-axis (Table 2).Moreover, the phytocompound has even been reported to improve cognitive and motor deficits in mouse studies of the same diseases (Schepici et al., 2020) (Table 2).

Pharmacological activation of NRF2: BACH1 inhibitors
BACH1 antagonizes NRF2 at the DNA binding site of antioxidant genes and thereby represses the expression of NRF2s target genes.In fact, nuclear exclusion of BACH1 has been reported to be necessary for the activation of neuroprotective pathways through its antioxidant target gene: HMOX-1, the rate-limiting enzyme in heme catabolism (Reichard et al., 2007).Despite the therapeutic potential of the NRF2-  Heme is BACH1s own endogenous ligand and its oxidized form, hemin, is the primary compound used to inhibit BACH1.An AD in vitro study demonstrated heme-mediated increase of the basal expression of NRF2 antioxidant target genes (Zhang et al., 2021) (Table 3).However, the development of heme as a safe therapeutic agent presents significant challenges, as it catalyzes the formation of ROS upon interaction with atmospheric oxygen, potentially leading to cellular damage and oxidative stress.Another compound that was identified as potent BACH1-inhibitor was the phytocannabinoid cannabidiol (CBD), which induces nuclear export and degradation of the transcription factor (Casares et al., 2020).In vitro models have confirmed CBDs antioxidant and anti-inflammatory properties, acting through HMOX-1 and inducing the downregulation of pro-inflammatory cytokines (Juknat et al., 2016;Duvigneau et al., 2020).In an AD mouse model, CBD treatment reduced neuroinflammation and facilitated neurogenesis in the hippocampus, although this was thought to be mainly mediated through PPARγ (Esposito et al., 2011) (Table 3).Recently, a study identified HPPE, a substituted benzimidazole, as BACH1 inhibitor coupled to activation of NRF2 (Ahuja et al., 2021).In MPTP-mice, HPPE treatment significantly blocked accumulation of oxidative stress markers, reduced neuro-inflammation and correspondingly, it increased neuroprotective genes (Ahuja et al., 2021) (Table 3).

Disease Experimental model Treatment regimen Major outcomes References
In conclusion, despite a limited number of studies, the targeting BACH1 has shown promise in achieving disease modification for NDD.

Molecular challenges in activating the NRF2 antioxidant response
Even though KEAP1 shows potential as a molecular target for the rescue of NRF2 defects, targeting KEAP1 for therapeutic purposes remains a challenge.First, KEAP1 has been shown to have complex interactions with various other proteins besides NRF2 (Hushpulian et al., 2021) and concerns about potential side effects arise from the diverse array of cellular processes covered by KEAP1 interactor proteins, involving DNA replication, cytoskeletal dynamics, transcription, and apoptosis.Secondly, small molecules designed to displace KEAP1 from NRF2 primarily target the Kelch domain in KEAP1.However, it is crucial to note that KEAP1 is only one member of a larger group of 42 Kelch-BTB proteins, each exerting essential roles in biological processes, primarily through substrate ubiquitination (Shi et al., 2019).Consequently, a small molecule targeting the Kelch domain of KEAP1 is highly likely to cross-react with the other proteins containing Kelch domains.Numerous studies on neurodegenerative diseases have shown that NRF2 stabilizes under chronic oxidative stress and inflammation but it still fails to increase the antioxidant genetic  program to the level needed to fight against the ongoing oxidative stress.One explanation for this is attributed to the feedback regulation within the pathway-continuous NRF2 activation triggers the cellular program to express NRF2 transcriptional repressors (Figures 4A,B).A study demonstrated that the induction of NRF2-mediated cytoprotective responses is diminished in cells from older adults, which is accompanied by an increase of its transcriptional repressor BACH1 (Zhou et al., 2018).Conversely, the expression of NRF2-target genes was shown to be enhanced in silenced BACH1 cells from older subjects (Zhang et al., 2019).Hence, BACH1 provides feedback mechanisms to counteract activation of NRF2 meaning that NRF2 protein stabilization is accompanied by an increase in expression levels of its own epigenetic repressor BACH1 (Zhou et al., 2018;Zhang et al., 2019).Indeed, upregulated BACH1 levels have been reported in post-mortem PD brains as well as in preclinical disease models and further associated with repression of the expression of NRF2 protective genes (Zhou et al., 2018;Ahuja et al., 2021).BACH1 knockout mice have been reported to be protected against MPTP-induced DA neurotoxicity and associated oxidative damage and neuroinflammation, which was substantiated by functional genomics data that demonstrated increased BACH1targeted pathways (Ahuja et al., 2021).Considering the above and BACH1s vital role in maintaining cellular redox homeostasis, the transcription factor presents a novel molecular target which intrinsically modulates the NRF2 pathway without breaking its negative-feedback loop (Figure 4C).

Novel drug modalities for NRF2 activation
Up to date, the only NRF2 activators in clinical use are DMF for MS (Rosito et al., 2020), and more recently Omaveloxolone, a semisynthetic terpenoid with potent NRF2-activating effects gained FDA approval in Februrary 2023 for the treatment of Friederich's ataxia (Boesch and Indelicato, 2024), a rare inherited neurodegenerative disease.Despite its recent approval, the mechanism of action of Omevaloxolone is still unknown, although it is thought to act through KEAP1 inhibition.
However, most NRF2 activators are of electrophilic nature and exhibit unpredictable poly-pharmacology.This was confirmed in one MS mouse model, which showed that DMF-mediated protection against acute inflammatory autoimmune encephalomyelitis was observed in both NRF2-proficient and NRF2-deficient animals (Schulze-Topphoff et al., 2016) and likewise, SFN has been demonstrated to have hundreds of molecular targets next to KEAP1 (Clulow et al., 2017).Due to that, many have failed to receive FDA approval.To overcome the problem of unknown and unpredictable off-target effects of electrophiles, small molecule protein-protein inhibitors (PPI) have been developed (Li et al., 2020).Those non-covalent compounds are highly specific and since they bind reversibly, they may exhibit a better safety profile (Abed et al., 2015).However, existing small molecules lack in vivo activity (Zhuang et al., 2017), potentially because they are highly polar and hardly able to penetrate the BBB.Due to their poor stability and low bioavailability, it is difficult to develop KEAP1-NRF2 PPIs as neurodegenerative drugs.
In contrast, RNA-based therapies have gained interest as a promising modality in treating neurological diseases (Holm et al., 2022) and they demonstrate advantages over conventional small molecule pharmaceuticals.Due to their mode of action being the direct modification, upregulation, or inhibition of transcripts (Juliano and Carver, 2015;Crooke, 2017), RNA-based drugs can be designed to modulate their therapeutic target specifically, rendering them highly valuable for pharmacologic interaction with biomolecules, or entire disease pathways, which are currently considered undruggable with small molecules or monoclonal antibodies.Hence, RNA therapeutics have great potential to be developed as personalized medicine, targeting heterogeneous characteristics of neurodegenerative diseases in patients individually.Since the only information required for their synthesis is the sequence of the target RNA (Kim et al., 2019), screening of drug lead candidates can be performed rapidly, and the availability of commercial vendors allows for a fast and cheap turnaround time.When RNA therapeutics are applied in the brain, they are usually delivered to the CNS through intrathecal injections, ensuring adequate drug levels in disease-relevant areas by circumventing the BBB (Holm et al., 2022).Although this has several advantages, it requires administration in primary care centers and long dosing intervals.However, chemical modifications have improved the drug-like characteristics of RNA modalities (Khvorova and Watts, 2017), rendering them safer and more stable in vivo, with half-lives extending over 6 months for Antisense Oligonucleotides (Scoles et al., 2019).Accordingly, RNA drug platforms represent a promising novel modality to make disease-modifying therapies for neurodegenerative diseases a reality.

Concluding remarks
The transcription factor NRF2 is central to the regulatory network governing cellular defenses against toxic and oxidative insults.When activated, this master regulator and its target genes attenuate numerous processes involved in neurodegeneration, including oxidative stress, neuroinflammation and mitochondrial dysfunction.In contrast to therapeutic interventions based on solely rectifying the pathology of proteinopathies, the stimulation of a pathway governing multiple disease processes holds the potential to modify disease dynamics and progression.Thus, targeting the NRF2-ARE axis can provide long-lasting and comprehensive neuronal protection, rendering it highly attractive for the treatment of neurodegenerative diseases.Presently, the plethora of identified NRF2 activators is of electrophilic nature and targets the reactive cysteine residues of KEAP1.Despite their potential to ameliorate neurodegeneration through upregulation of NRF2 and its target genes, as shown by numerous pre-clinical studies, their molecular off-target effects and concomitant unpredictable side-effects pose an obstacle for clinical trials and approval.On the other hand, PPIs that have evolved to overcome the drawbacks of covalent modifiers lack bioavailability and are currently not applicable for the treatment of neurological disorders.A more recently established target in the antioxidant pathway is BACH1, which serves as a negative regulator of NRF2.By acting as a physiological repressor of NRF2 target genes at the ARE-binding site, knockdown or nuclear exclusion of BACH1 holds promising potential for reactivating the downregulated activity of NRF2 associated with disease.Future challenges to overcome in the development of NRF2 activators as disease modifying treatments include the establishment of compounds with good pharmacokinetic and pharmacodynamic profiles that ensure sustained, brain wide NRF2 activation.In light of this, RNAbased therapies hold greater therapeutic potential than current pharmaceutical designs, and offer the possibility to revolutionize the therapeutic landscape of neurodegenerative diseases.

FIGURE 4
FIGURE 4Molecular targets within the NRF2 pathway (A) In oxidative stress conditions, NRF2 signaling is hampered by ongoing proteasome-dependent degradation in the cytoplasm through KEAP1, and antagonistic repression of NRF2's DNA-binding site in the nucleus by BACH1.(B) Most NRF2 activators modify or replace KEAP1 to facilitate NRF2's translocation into the nucleus, however, higher levels of NRF2 are accompanied by an increased negative feedback response, and heightened BACH1 levels in the nucleus impede the full antioxidant gene expression by occupying the ARE binding site.(C) Downregulating transcriptional inhibitor BACH1 allows the induction of NRF2-induced antioxidant genes without activating negative feedback regulators.

TABLE 1
DMF's ability to alleviate pathological hallmarks of AD, PD and ALS in pre-clinical models in the context of Nrf2 activation.This table summarizes the impact of DMF on key pathological features observed in pre-clinical models of AD, PD, and ALS.The data highlights DMF's ability to modulate disease hallmarks through the activation of the NRF2 pathway.Additionally, the table details the extent of neuroprotection, oxidative stress reduction, and antiinflammatory effects achieved by DMF treatment in the various disease models.

TABLE 2
Sulforaphane provides neuroprotection in NDD models through activation of the antioxidant NRF2/ARE pathway This table illustrates the neuroprotective properties of SFN in various pre-clinical models of AD, PD and MS.Key outcomes include the reduction of oxidative stress, mitigation of neuronal damage, and enhancement of cellular defense mechanisms.The table also highlights the improvements in cognitive and motor functions observed in vivo disease models following SFN treatment.

TABLE 2 (
Continued) Sulforaphane provides neuroprotection in NDD models through activation of the antioxidant NRF2/ARE pathway This table illustrates the neuroprotective properties of SFN in various pre-clinical models of AD, PD and MS.Key outcomes include the reduction of oxidative stress, mitigation of neuronal damage, and enhancement of cellular defense mechanisms.The table also highlights the improvements in cognitive and motor functions observed in vivo disease models following SFN treatment.

TABLE 3
BACH1 inhibitors induce the NRF2 pathway in neurodegenerative conditions.This table details how BACH1 inhibiting compounds leads to the activation of NRF2, promoting antioxidant responses and providing neuroprotection.The table includes data from pre-clinical models of AD and PD, showing reductions in oxidative stress, decreased neuronal loss, and improvements in disease-related symptoms following treatment with BACH1 inhibitors.